1. Field of the Invention
The present invention relates to biological structures designed to induce apoptosis in HIV-1 infected cells, and more specifically to recombinant human serum transferrins designed to introduce apoptosis-inducing peptides into HIV-1 infected cells.
2. Description of the Prior Art
The preservation of equilibrium in a multicellular organism, the process of homeostasis, requires a delicate balance between cell proliferation and cell death. In a well developed, adult organism the maintenance and renewal of many specialized cell lines is a matter of continuing importance. Therefore, cell proliferation and in particular its tight checks and balances necessary for optimal adult health has been a process of keen interest to those involved in biological research. Accordingly, the study of infectious diseases and more recently the focused inspection of mechanisms associated with cancer have followed these adult self-preservation paths, expanding our knowledge of the many factors involved in cell proliferationxe2x80x94where some of the more recent research has elucidated the importance of growth factors, proto-oncogenes, and tumor suppressors such as p53.
Over the whole life cycle the viability of an organism is maintained by a continuous balance between cell proliferation and cell death by necrosis or apoptosis. While the necessity of cell suicide in an organized biological system has been well appreciated, it is only recently that it was given substantial research attention as a therapeutic mechanism.
For example, the study of Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV) has revealed that uninfected CD4+ T cells are induced to undergo apoptosis by adjacent HIV-infected cells. It appears that the survival strategy of this virus depends in part on causing mass suicide in the ranks of its primary immunological opponent, the T lymphocyte, while remaining concealed in the suicide inducer, the infected cell. See, e.g., Meyaard, L. et al. (1992) Science 257:217; Groux, H. et al. (1992) J. Exp. Med. 175:331; Banda, N. K. et al. (1992) J. Exp. Med. 176:1099. Since apoptosis, unlike necrotic cell death, does not normally evoke inflammation or a strong immune response, this viral stratagem is particularly successful; the disease remains essentially symptom free until opportunistic infections are allowed to take hold as a result of depleted T cell ranks.
The opposing therapeutic stratagems need equal brilliance. Following the clandestine path of this virus, the use of apoptosis induction as a therapeutic tool is attractive as it can avoid potentially harmful inflammatory and immune system responses. The insidious nature of the HIV process, its current prevalence, and its essentially asymptomatic onset are best opposed by equally asymptomatic response mechanisms in any therapeutic regimen. Accordingly, therapeutic mechanisms that induce apoptosis in HIV infected cells are universally desired, and it is one such mechanism that is disclosed herein.
Those in the field will appreciate that recent (1999) estimates by the World Health Organization (WHO) indicate that there are no less than 33.6 million people living with HIV/AIDS. Recently it has been observed and reported that the combination of simultaneous protease and reverse transcriptase inhibitors does not confer resistance to HIV. See Michael, N. and Moore, J. (1999) Nature Medicine 5:7, 740-741. The infective nature of this disease offers only somber future statistics. Accordingly, various new drugs to combat this epidemic have been proposed, including those based on cleavage by HIV-1 protease, such as the toxic pro-drugs activated inside the infected cell (the Trojan Horse strategy), peptide drugs and others.
At the same time the study of natural transport proteins has revealed the unique attributes of the human serum transferrin as a delivery mechanism, and its concurrent convenience for recombinant manipulation. For example, U.S. Pat. No. 5,986,067 to Funk et al. (1999) teaches the usefulness of recombinant transferrin proteins as vehicles for metal chelation while Ali, S. A. et al. (1999) J. Biol. Chem. 274:34, 24066-24073 describe the insertion of a peptide sequence into the N-terminal lobe of human serum transferrin via cassette mutagenesis and PCR-ligation-PCR mutagenesis techniques.
When Ali et al. inserted a peptide into the N-terminal lobe of human serum transferrin (HST), they demonstrated that the recombinant HST retained its native functions, including mediation of iron transport and uptake into cells. Human serum transferrin is a monomeric glycoprotein that binds tightly and reversibly to two ferric ions together with two bicarbonate co-ions. The roles of HST are 1) the regulation of the availability of free iron in body fluids and 2) mediation of the transport and uptake of iron into cells via receptor-mediated endocytosis. Even though all living cells need iron, only those cells which have high iron requirement (i.e. HIV-infected cells and cancer cells) express large numbers of transferrin receptors. See lacopetta et al. (1982) Biochem. Biophys. Acta. 687:204-210. Significantly, HIV-1 replication has been shown to induce up-regulation of HST receptor expression, and therefore HIV-1 infected cells are particularly susceptible to an HST-directed therapy. The use of recombinant human serum transferrin is therefore uniquely suitable for the process disclosed herein.
An essential step in the life cycle of human immunodeficiency virus type-1 (HIV-1) is the processing of the Gag and Gag-Pol polyproteins which is performed by the virus encoded HIV-1 protease. The cleavage of viral polyproteins by HIV-1 protease results in structural and catalytic proteins required for both viral maturation and infection, and therefore HIV-1 protease has been a target of anti-AIDS drugs. See Friedler, A. et al. (1999) J. Mol. Biol. 287:93-101. One novel approach that has been recently examined is the use of toxic prodrugs which are cleaved selectively in cells infected by HIV-1xe2x80x94often called the xe2x80x9cTrojan Horsexe2x80x9d strategy. A chimeric Vpr protein (an auxiliary HIV-1 gene product) which contained at its C-terminus nine HIV-1 protease cleavage sites was shown to completely abolish viral infectivity as cleavage of the chimeric protein interfered with the processing of viral precursor proteinsxe2x80x94leading to the production of incompletely processed noninfectious virus particles. See Serio, D. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3346-3351. The current invention described herein combines the Trojan Horse strategy with recent knowledge of the existence of peptides which cause cell suicide. The proposed recombinant human serum transferrins would inhibit HIV-1 by acting as competitive substrates for HIV-1 protease and in so doing would also selectively release xe2x80x9ccell suicidexe2x80x9d0 peptides in the HIV-1 infected cell. Therefore, the proposed invention endeavors to not only inhibit the virulence of HIV-1 but also to eradicate those cells infected by HIV-1.
Accordingly it is the general purpose and objective of the present invention to design a recombinant mammalian protein directed at seizing the normal process of apoptosis or xe2x80x9ccell suicidexe2x80x9d as a therapeutic stratagem.
Further objects of the invention are to create various recombinant human serum transferrin proteins containing one of several peptides flanked by two HIV-1 protease cleavage sites and possessing an apoptosis-inducing motif.
Other objectives of the invention are to provide a modification of a protein structure generally functional in basic biological processes whereby the protein modification includes apoptotic peptide segments inserted between cleavage sites selected for a targeted infection.
Yet further objectives of the invention are to create recombinant modifications of a natural transport protein so as to conform it to induce cell apoptosis in an HIV-1 infected cell.
Additional objectives of the invention are to insert an apoptotic mammalian peptide which may be released from the designed recombinant mammalian protein in an infected cell via cleavage by the HIV-1 protease enzyme.
In each of the foregoing objects the invention aims to create such modifications in a way that will not compromise the biological roles of the human serum transferrin protein.
Briefly, these and other objects are accomplished within the present invention by modifying a mammalian protein, like the human serum transferrin (HST) protein, with an inserted peptide sequence flanked at both ends by cleavage sites. The proposed peptide insert contains a motif known to induce apoptosis in cells and the cleavage sites are specific for the viral protease of HIV-1. The delivery of such recombinant transferrin into an HIV-1 infected cell may result in the release of the peptide which can then induce apoptosis.
The peptides designed in accordance with the present invention for insertion into surface exposed loops of the N-terminal lobe of human serum transferrin are: SEQ ID No: 1 VSQNYVIVLRGDVSQNYVIVL SEQ ID No: 2 VSQNYVIVLRGDSVSQNYVIVL SEQ ID No: 3 VSQNYVIVLGRGDNPVSQNYVIVL, SEQ ID No: 4 and VSQNYVIVLGRGDSPVSQNYVIVL. These peptide inserts include in each instance an apoptosis inducing peptide containing the RGD motif (indicated by bold, underlined script) flanked by two modified p17/p24 HIV-1 protease cleavage sites.
When delivered to the infected cell, the cleavage of the loop inserted sequences by the HIV-1 protease results in the release of the central RGD-containing peptide sequences. Peptides containing the RGD motif (arginine, glycine, aspartic acid) have been recently shown to induce cell apoptosis even in small concentrations (as low as 250 micromolar). See Buckley, C. et al. (1999) Nature 397:534-39. Buckley et al. demonstrates that peptides containing the RGD motif (the minimal sequence required to induce apoptosis was the RGD motif by itself) induced rapid apoptosis in resting (G0) peripheral blood T cells, CD4-positive T-cell lines, leukaemic T-cell lines (Jurkat and Molt-4 cells), B cells transformed by Epstein-Barr virus (LCL cells), and the erythroleukaemic cell line K562. The current hypothesis is that the RGD-containing peptide directly activates caspase-3, the apoptotic point of no return. Caspase-3 is a an apoptotic zymogen whose activation allows it to cleave the inhibitor of caspase-activated DNAase, leading to cell suicide. It is of particular significance to this invention that the RGD peptide motif has been shown to induce apoptosis in CD4-positive T cells, as the CD4-positive T helper cell is the primary target of the lymphotropic HIV virus; see Kuby, J. Immunology. 3rd Edition. W. H. Freeman and Co., New York, 1997.
The sequences flanking the RGD-containing peptides (bold, underlined) above are modified p17/p24 HIV-1 protease cleavage sites (VSQNYVIVL). The natural p17/p24 HIV-1 protease cleavage site is VSQNYPIVL, and HIV-1 protease cleaves at the YP junction. The modified p17/p24 HIV-1 protease cleavage sites (VSQNYVIVL) used in the designed peptides have a proline to valine substitution in the P1xe2x80x2 position of the cleavage site. This modification was used so that the peptides released following cleavage by HIV-1 protease have a valine (V) N-terminal terminal amino acid which is stabilizing according to the N-end rule; the mammalian N-end rule associates the intracellular half-life of a protein with its N-terminal amino acid. Cleavage of the modified p17/p24 HIV-1 protease cleavage sites occurs at the YV junction, and the substitution of valine for proline has been shown to only have a minor effect on cleavage by HIV-1 protease. See Falnes et al. (1999) Biochem. J. 343:199-207. This modification substantially improves the stability of the released apoptosis-inducing peptides so that they are more likely to activate caspase-3 and the cell suicide pathway.
Those in the art will appreciate that human serum transferrin, a monomeric glycoprotein that binds tightly and reversibly to two ferric ions together with two bicarbonate co-ions, regulates the availability of free iron in body fluids, and also mediates the transport and cell uptake of iron by receptor-mediated endocytosis. See Huebers, H. et al. (1981) Proc. Natl. Acad. Sci. USA 78:1, 621-625; Jeffrey P. D. et al.(1998) Biochemistry 37:13978-13986; Ali, S. A et al. (1999) J. Biol. Chem. 274:34, 24066-24073. By this process, the transferrin molecule binds to the transferrin receptor on the membrane of cells and the complex is subsequently enveloped by the cell. Significantly, HIV replication has been shown to induce upregulation of HST receptor expression since HIV-infected cells exhibit a higher need for iron. See Levy, J. A. (1993) Microbiol. Rev. 57:183-289. Even more significantly, while conjugating peptides to human serum transferrin has been shown to induce an antibody immune response, hiding peptides within the loops of transferrin has not been associated with detectable antibody production. See Ali et al. (1999) FEBS Letters 459:230-232. Thus, human serum tranferrin provides an attractive pathway for the delivery of engineered peptides into HIV-infected cells as it takes advantage of a pre-evolved cell entry mechanism, exercises selectivity for HIV-infected cells which possess a greater number of transferrin receptors, and minimizes immunogenicity which often complicates the use of peptide drugs.
The efficacy of this mechanism is further enhanced by selecting insertion sites that are removed from the transferrin C-terminal lobe, known to be important for iron and bicarbonate binding. The C-terminal lobe of transferrin also possesses the primary sites for HST receptor recognition. See Zak et. al., J. Biol. Chem., 269:10, 7110-7114. Stability of the peptides once released into the cytoplasm is improved by including two modified p17/p24 HIV-1 protease cleavage sites in substitution for the natural p17/p24 cleavage sequence VSQNYPIVL. In this manner the released peptide conforms with the stabilizing N-end rule, and the peptide can be inserted into the N-terminal lobe surface exposed loops of transferrin, which are removed from the iron transport machinery. Surface exposed loops of the HST protein are the chosen targets of peptide insertion due to their flexibility and therefore resulting ability to tolerate insertions without significantly distorting the overall tertiary or three-dimensional spatial structure of the protein. See Finkelstein, A. V. (1997) Curr. Opin. Struct. Biol. 7:60-71. Furthermore, surface exposed loops are chosen so that peptide sequences can be exposed to HIV-1 protease recognition and cleavage.